The Role of Green Tea Catechin Epigallocatechin Gallate (EGCG) and Mammalian Target of Rapamycin (mTOR) Inhibitor PP242 (Torkinib) in the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition that has physical and psychological consequences for patients. SCI is accompanied by scar formation and systemic inflammatory response leading to an intense degree of functional loss. The catechin, epigallocatechin gallate (EGCG), an active compound found in green tea, holds neuroprotective features and is known for its anti-inflammatory potential. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two functionally distinct complexes termed mTOR complex 1 and 2 (mTORC1; mTORC2). Inhibition of mTORC1 by rapamycin causes neuroprotection, leading to partial recovery from SCI. In this study the effects of EGCG, PP242 (an inhibitor of both complexes of mTOR), and a combination of EGCG and PP242 in SCI have been examined. It has been found that both EGCG and PP242 significantly improved sensory/motor functions following SCI. However, EGCG appeared to be more effective (BBB motor test, from 2 to 8 weeks after SCI, p = 0.019, p = 0.007, p = 0.006, p = 0.006, p = 0.05, p = 0.006, and p = 0.003, respectively). The only exception was the Von Frey test, where EGCG was ineffective, while mTOR inhibition by PP242, as well as PP242 in combination with EGCG, significantly reduced withdrawal latency starting from week three (combinatorial therapy (EGCG + PP242) vs. control at 3, 5, and 7 weeks, p = 0.011, p = 0.007, and p = 0.05, respectively). It has been found that EGCG was as effective as PP242 in suppressing mTOR signaling pathways, as evidenced by a reduction in phosphorylated S6 expression (PP242 (t-test, p < 0.0001) or EGCG (t-test, p = 0.0002)). These results demonstrate that EGCG and PP242 effectively suppress mTOR pathways, resulting in recovery from SCI in rats, and that EGCG acts via suppressing mTOR pathways.

Involvement of mTOR Pathways in Recovery from Spinal Cord Injury by Modulation of Autophagy and Immune Response.

Traumatic spinal cord injury (SCI) is untreatable and remains the leading cause of disability. Neuroprotection and recovery after SCI can be partially achieved by rapamycin (RAPA) treatment, an inhibitor of mTORC1, complex 1 of the mammalian target of rapamycin (mTOR) pathway. However, mechanisms regulated by the mTOR pathway are not only controlled by mTORC1, but also by a second mTOR complex (mTORC2). Second-generation inhibitor, pp242, inhibits both mTORC1 and mtORC2, which led us to explore its therapeutic potential after SCI and compare it to RAPA treatment. In a rat balloon-compression model of SCI, the effect of daily RAPA (5 mg/kg; IP) and pp242 (5 mg/kg; IP) treatment on inflammatory responses and autophagy was observed. We demonstrated inhibition of the mTOR pathway after SCI through analysis of p-S6, p-Akt, and p-4E-BP1 levels. Several proinflammatory cytokines were elevated in pp242-treated rats, while RAPA treatment led to a decrease in proinflammatory cytokines. Both RAPA and pp242 treatments caused an upregulation of LC3B and led to improved functional and structural recovery in acute SCI compared to the controls, however, a greater axonal sprouting was seen following RAPA treatment. These results suggest that dual mTOR inhibition by pp242 after SCI induces distinct mechanisms and leads to recovery somewhat inferior to that following RAPA treatment.

Metabolic Changes in Focal Brain Ischemia in Rats Treated With Human Induced Pluripotent Stem Cell-Derived Neural Precursors Confirm the Beneficial Effect of Transplanted Cells.

There is currently no treatment for restoring lost neurological function after stroke. A growing number of studies have highlighted the potential of stem cells. However, the mechanisms underlying their beneficial effect have yet to be explored in sufficient detail. In this study, we transplanted human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) in rat temporary middle cerebral artery occlusion (MCAO) model. Using magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) we monitored the effect of cells and assessed lesion volume and metabolite changes in the brain. We monitored concentration changes of myo-inositol (Ins), Taurine (Tau), Glycerophosphocholine+Phosphocholine (GPC+PCh), N-acetyl-aspartate+N-acetyl-aspartyl-glutamate (NAA+NAAG), Creatine+Phosphocreatine (Cr+PCr), and Glutamate+Glutamine (Glu+Gln) in the brains of control and iPSC-NP-transplanted rats. Based on initial lesion size, animals were divided into small lesion and big lesion groups. In the small lesion control group (SCL), lesion size after 4 months was three times smaller than initial measurements. In the small lesion iPSC-NP-treated group, lesion volume decreased after 1 month and then increased after 4 months. Although animals with small lesions significantly improved their motor skills after iPSC-NP transplantation, animals with big lesions showed no improvement. However, our MRI data demonstrate that in the big lesion iPSC-NP-treated (BTL) group, lesion size increased only up until 1 month after MCAO induction and then decreased. In contrast, in the big lesion control group, lesion size increased throughout the whole experiment. Significantly higher concentrations of Ins, Tau, GPC+PCh, NAA+NAAG, Cr+PCr, and Glu+Gln were found in in contralateral hemisphere in BTL animals 4 months after cell injection. Lesion volume decreased at this time point. Spectroscopic results of metabolite concentrations in lesion correlated with volumetric measurements of lesion, with the highest negative correlation observed for NAA+NAAG. Altogether, our results suggest that iPSC-NP transplantation decreases lesion volume and regulates metabolite concentrations within the normal range expected in healthy tissue. Further research into the ability of iPSC-NPs to differentiate into tissue-specific neurons and its effect on the long-term restoration of lesioned tissue is necessary.

Transplantation of neural precursors generated from spinal progenitor cells reduces inflammation in spinal cord injury via NF-κB pathway inhibition.

BACKGROUND: Traumatic spinal cord injury (SCI) triggers a chain of events that is accompanied by an inflammatory reaction leading to necrotic cell death at the core of the injury site, which is restricted by astrogliosis and apoptotic cell death in the surrounding areas. Activation of nuclear factor-κB (NF-κB) signaling pathway has been shown to be associated with inflammatory response induced by SCI. Here, we elucidate the pattern of activation of NF-κB in the pathology of SCI in rats and investigate the effect of transplantation of spinal neural precursors (SPC-01) on its activity and related astrogliosis. METHODS: Using a rat compression model of SCI, we transplanted SPC-01 cells or injected saline into the lesion 7 days after SCI induction. Paraffin-embedded sections were used to assess p65 NF-κB nuclear translocation at days 1, 3, 7, 10, 14, and 28 and to determine levels of glial scaring, white and gray matter preservation, and cavity size at day 28 after SCI. Additionally, levels of p65 phosphorylated at Serine536 were determined 10, 14, and 28 days after SCI as well as levels of locally secreted TNF-α. RESULTS: We determined a bimodal activation pattern of canonical p65 NF-κB signaling pathway in the pathology of SCI with peaks at 3 and 28 days after injury induction. Transplantation of SCI-01 cells resulted in significant downregulation of TNF-α production at 10 and 14 days after SCI and in strong inhibition of p65 NF-κB activity at 28 days after SCI, mainly in the gray matter. Moreover, reduced formation of glial scar was found in SPC-01-transplanted rats along with enhanced gray matter preservation and reduced cavity size. CONCLUSIONS: The results of this study demonstrate strong immunomodulatory properties of SPC-01 cells based on inhibition of a major signaling pathway. Canonical NF-κB pathway activation underlines much of the immune response after SCI including cytokine, chemokine, and apoptosis-related factor production as well as immune cell activation and infiltration. Reduced inflammation may have led to observed tissue sparing. Additionally, such immune response modulation could have impacted astrocyte activation resulting in a reduced glial scar.

Modified Methacrylate Hydrogels Improve Tissue Repair after Spinal Cord Injury.

Methacrylate hydrogels have been extensively used as bridging scaffolds in experimental spinal cord injury (SCI) research. As synthetic materials, they can be modified, which leads to improved bridging of the lesion. Fibronectin, a glycoprotein of the extracellular matrix produced by reactive astrocytes after SCI, is known to promote cell adhesion. We implanted 3 methacrylate hydrogels: a scaffold based on hydroxypropylmethacrylamid (HPMA), 2-hydroxyethylmethacrylate (HEMA) and a HEMA hydrogel with an attached fibronectin (HEMA-Fn) in an experimental model of acute SCI in rats. The animals underwent functional evaluation once a week and the spinal cords were histologically assessed 3 months after hydrogel implantation. We found that both the HPMA and the HEMA-Fn hydrogel scaffolds lead to partial sensory improvement compared to control animals and animals treated with plain HEMA scaffold. The HPMA scaffold showed an increased connective tissue infiltration compared to plain HEMA hydrogels. There was a tendency towards connective tissue infiltration and higher blood vessel ingrowth in the HEMA-Fn scaffold. HPMA hydrogels showed a significantly increased axonal ingrowth compared to HEMA-Fn and plain HEMA; while there were some neurofilaments in the peripheral as well as the central region of the HEMA-Fn scaffold, no neurofilaments were found in plain HEMA hydrogels. In conclusion, HPMA hydrogel as well as the HEMA-Fn scaffold showed better bridging qualities compared to the plain HEMA hydrogel, which resulted in very limited partial sensory improvement.

Does combined therapy of curcumin and epigallocatechin gallate have a synergistic neuroprotective effect against spinal cord injury?

Systematic inflammatory response after spinal cord injury (SCI) is one of the factors leading to lesion development and a profound degree of functional loss. Anti-inflammatory compounds, such as curcumin and epigallocatechin gallate (EGCG) are known for their neuroprotective effects. In this study, we investigated the effect of combined therapy of curcumin and EGCG in a rat model of acute SCI induced by balloon compression. Immediately after SCI, rats received curcumin, EGCG, curcumin + EGCG or saline [daily intraperitoneal doses (curcumin, 6 mg/kg; EGCG 17 mg/kg)] and weekly intramuscular doses (curcumin, 60 mg/kg; EGCG 17 mg/kg)] for 28 days. Rats were evaluated using behavioral tests (the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test, flat beam test). Spinal cord tissue was analyzed using histological methods (Luxol Blue-cresyl violet staining) and immunohistochemistry (anti-glial fibrillary acidic protein, anti-growth associated protein 43). Cytokine levels (interleukin-1ß, interleukin-4, interleukin-2, interleukin-6, macrophage inflammatory protein 1-alpha, and RANTES) were measured using Luminex assay. Quantitative polymerase chain reaction was performed to determine the relative expression of genes (Sort1, Fgf2, Irf5, Mrc1, Olig2, Casp3, Gap43, Gfap, Vegf, NfκB, Cntf) related to regenerative processes in injured spinal cord. We found that all treatments displayed significant behavioral recovery, with no obvious synergistic effect after combined therapy of curcumin and ECGC. Curcumin and EGCG alone or in combination increased axonal sprouting, decreased glial scar formation, and altered the levels of macrophage inflammatory protein 1-alpha, interleukin-1ß, interleukin-4 and interleukin-6 cytokines. These results imply that although the expected synergistic response of this combined therapy was less obvious, aspects of tissue regeneration and immune responses in severe SCI were evident.

A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines.

Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. Behavior recovery was monitored by BBB, plantar, rotarod and flat-beam tests. The levels of inflammatory cytokines were determined on days 1, 3, 7, 10 and 14 after SCI. Additionally, NF-κB pathway activity was evaluated. The results demonstrated that EGCG-treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL-6, IL2, MIP1α and RANTES levels on days 1 and 3, and an upregulation of IL-4, IL-12p70 and TNFα 1 day following SCI in EGCG-treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF-κB dimer, and therefore canonical NF-κB pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG-treated rats. Further, EGCG influenced expression of M1 and M2 macrophage markers. Our results have demonstrated a therapeutic value of EGCG in SCI, as observed by better behavioral performance measured by flat beam test, modulation of inflammatory cytokines and induction of higher axonal sprouting.

The Osteogenic Potential of Human Nondifferentiated and Pre-differentiated Mesenchymal Stem Cells Combined with an Osteoconductive Scaffold - Early Stage Healing.

Despite the huge research into stem cells and their regenerative properties for bone healing, there are still unanswered questions including the recipient's respond to the presence of the stem cells, the fate of stem cells inside the bone defect and the possible advantage in utilizing pre-differentiated cells. To address these problems, we used human multipotent mesenchymal stromal/stem cells (MSCs), GMP Grade, in a rat model of bone formation. In a "bioreactor concept" approach seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 2 × 106 MSCs, seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 1 × 106 predifferentiated osteoblasts and 1 × 106 pre-differentiated endothelial cells and 14 Wistar rats were implanted with 0.2 g of synthetic bone scaffold without seeded cells into an intramuscular pocket on the left side of their back. The right side of each rat was used as a control, and 0.2 g of synthetic bone scaffold was implanted into the intramuscular pocket alone. To see the early stage healing the samples were harvested 14 days after the implantation, MSCs were detected by positive DAPI and MTCO2 staining in 43% of all the samples implanted with MSCs, and no inflammation signs were present in any implanted animal. New vessels could be found in both groups implanted with MSCs, but not in the control group of animals. However, hematoxylin-eosin staining could not detect newly created bone within the implant in any of the groups. These results were in line with COLL1 staining, where we could detect positive staining only in three cases, all of which were implanted with un-differentiated MSCs. According to our findings, there were no benefits of using the pre-differentiated of MSC.

A Comparative Study of Three Different Types of Stem Cells for Treatment of Rat Spinal Cord Injury.

Three different sources of human stem cells-bone marrow-derived mesenchymal stem cells (BM-MSCs), neural progenitors (NPs) derived from immortalized spinal fetal cell line (SPC-01), and induced pluripotent stem cells (iPSCs)-were compared in the treatment of a balloon-induced spinal cord compression lesion in rats. One week after lesioning, the rats received either BM-MSCs (intrathecally) or NPs (SPC-01 cells or iPSC-NPs, both intraspinally), or saline. The rats were assessed for their locomotor skills (BBB, flat beam test, and rotarod). Morphometric analyses of spared white and gray matter, axonal sprouting, and glial scar formation, as well as qPCR and Luminex assay, were conducted to detect endogenous gene expression, while inflammatory cytokine levels were performed to evaluate the host tissue response to stem cell therapy. The highest locomotor recovery was observed in iPSC-NP-grafted animals, which also displayed the highest amount of preserved white and gray matter. Grafted iPSC-NPs and SPC-01 cells significantly increased the number of growth-associated protein 43 (GAP43+) axons, reduced astrogliosis, downregulated Casp3 expression, and increased IL-6 and IL-12 levels. hMSCs transiently decreased levels of inflammatory IL-2 and TNF-α. These findings correlate with the short survival of hMSCs, while NPs survived for 2 months and matured slowly into glia- and tissue-specific neuronal precursors. SPC-01 cells differentiated more in astroglial phenotypes with a dense structure of the implant, whereas iPSC-NPs displayed a more neuronal phenotype with a loose structure of the graft. We concluded that the BBB scores of iPSC-NP- and hMSC-injected rats were superior to the SPC-01-treated group. The iPSC-NP treatment of spinal cord injury (SCI) provided the highest recovery of locomotor function due to robust graft survival and its effect on tissue sparing, reduction of glial scarring, and increased axonal sprouting.

The distribution of lymphoid cells in the small intestine of germ-free and conventional piglets.

Porcine ileum is populated with a high proportion of B cells but previous studies have shown that they are not developed there. While B cells prevail in the ileum even in germ-free animals, microbial colonization is a major factor that causes even a greater prevalence of B cells in the ileum and further differential representation of lymphoid cells throughout small intestine. Analysis of lymphoid subpopulations showed that the effector cells appear only after colonization. These include class-switched IgM(+)IgA(+) B cells, primed CD2(-)CD21(+) B cells, antibody-producing/memory CD2(+)CD21(-) B cells, and effector/memory CD4(+)CD8(+) αß Th cells. While colonization resulted in a uniform distribution of effector cells throughout the gut, it caused a decrease in the frequency of cytotoxic αß and CD2(+)CD8(+) γδ T cells. These results suggest that the ileum is a site where naive B cells expand presumably to increase antibody repertoire but the entire small intestine is immunofunctionally comparable.

The Anti-Inflammatory Compound Curcumin Enhances Locomotor and Sensory Recovery after Spinal Cord Injury in Rats by Immunomodulation.

Well known for its anti-oxidative and anti-inflammation properties, curcumin is a polyphenol found in the rhizome of Curcuma longa. In this study, we evaluated the effects of curcumin on behavioral recovery, glial scar formation, tissue preservation, axonal sprouting, and inflammation after spinal cord injury (SCI) in male Wistar rats. The rats were randomized into two groups following a balloon compression injury at the level of T9-T10 of the spinal cord, namely vehicle- or curcumin-treated. Curcumin was applied locally on the surface of the injured spinal cord immediately following injury and then given intraperitoneally daily; the control rats were treated with vehicle in the same manner. Curcumin treatment improved behavioral recovery within the first week following SCI as evidenced by improved Basso, Beattie, and Bresnahan (BBB) test and plantar scores, representing locomotor and sensory performance, respectively. Furthermore, curcumin treatment decreased glial scar formation by decreasing the levels of MIP1α, IL-2, and RANTES production and by decreasing NF-κB activity. These results, therefore, demonstrate that curcumin has a profound anti-inflammatory therapeutic potential in the treatment of spinal cord injury, especially when given immediately after the injury.

Human mesenchymal stem cells modulate inflammatory cytokines after spinal cord injury in rat.

Transplantation of mesenchymal stem cells (MSC) improves functional recovery in experimental models of spinal cord injury (SCI); however, the mechanisms underlying this effect are not completely understood. We investigated the effect of intrathecal implantation of human MSC on functional recovery, astrogliosis and levels of inflammatory cytokines in rats using balloon-induced spinal cord compression lesions. Transplanted cells did not survive at the lesion site of the spinal cord; however, functional recovery was enhanced in the MSC-treated group as was confirmed by the Basso, Beattie, and Bresnahan (BBB) and the flat beam test. Morphometric analysis showed a significantly higher amount of remaining white matter in the cranial part of the lesioned spinal cords. Immunohistochemical analysis of the lesions indicated the rearrangement of the glial scar in MSC-treated animals. Real-time PCR analysis revealed an increased expression of Irf5, Mrc1, Fgf2, Gap43 and Gfap. Transplantation of MSCs into a lesioned spinal cord reduced TNFα, IL-4, IL-1ß, IL-2, IL-6 and IL-12 and increased the levels of MIP-1α and RANTES when compared to saline-treated controls. Intrathecal implantation of MSCs reduces the inflammatory reaction and apoptosis, improves functional recovery and modulates glial scar formation after SCI, regardless of cell survival. Therefore, repeated applications may prolong the beneficial effects induced by MSC application.

Ileal Peyer's patches are not necessary for systemic B cell development and maintenance and do not contribute significantly to the overall B cell pool in swine.

Based on studies of sheep, ileal Peyer's patches (IPP) have been regarded as a type of primary lymphoid tissue similar to the bursa of Fabricius in chicken. Because bursectomy results in B cell deficiency, we wondered whether resection of the IPP of piglets would have a similar effect. Comparison of IPP-resected, surgical shams and untreated germ-free piglets, all of which were later colonized with a defined commensal flora, demonstrated that resection of the IPP did not alter the level and phenotype of B and T cells in lymphoid tissues and the blood 10 wk after surgery. Additionally, colonization of IPP caused a shift from the fetal type of lymphocyte distribution to the adult type that is characterized by prevalence of B cells, with many of them representing IgA(+) switched B cells or displaying a more mature CD2(-)CD21(+) and CD2(-)CD21(-) phenotype. Moreover, colonization leads to appearance of effector CD4(+)CD8(+) αß T helper and CD2(+)CD8(-) γδ T cells. Comparison of germ-free with colonized pigs and experiments utilizing surgical transposition of jejunal Peyer's patch into terminal ileum or construction of isolated ileal loops indicated that lymphocyte development in IPP is dependent on colonization. Although our studies confirmed higher mitotic and apoptotic rates in IPP, they failed to identify any cell populations that resemble developing B lineage cells in the bone marrow. These results indicate that porcine IPP are not required for systemic B cell generation or maintenance, but they are secondary lymphoid tissue that appears important in immune responses to colonizing bacteria.